A barrier to simplified diagnostic testing is that current clinical chemistry technologies require significant sample preparation and handling for the analysis of complex biological samples. Sample preparation is a major bottleneck in diagnostics. Indicator fluorophores for specific biomarkers capable of functioning directly in an analyte's medium (e.g., blood, urine) without sample handling or separation steps would require fewer manipulations, thereby producing quicker results and reducing potential health hazards due to sample handling. However, surprisingly little progress has been made developing such fluorophores. This is due at least partially to the relative lack of long wavelength probes.
There are relatively few classes of near infrared (NIR) active dyes, or fluorophores, that are routinely used, and only one NIR dye is currently approved for clinical use. Advantages of NIR dyes include minimal interfering absorption and fluorescence from biological samples, inexpensive laser diode excitation, and reduced scattering and enhanced tissue penetration depth. However, there are only relatively few classes of such dyes readily available. These include the phthalocyanines, cyanine dyes and squaraine dyes. Each class of dye has inherent strengths and limitations. For example, almost all the established groups of long-wavelength fluorophores have very small Stokes shifts (i.e., emission-excitation wavelength differences), e.g., 10 nm (Miller, Springer Ser. Fluoresc., 2008, 5, 147-162). If used in conjunction with a relatively broad band light source, such as an LED, there may be significant scattered light background signal, producing a poor signal:noise ratio.
Previous research has investigated red-shifting xanthene dyes for biodiagnostics and imaging applications. Long-wavelength, xanthene-based dyes have been used in cellular imaging applications. However, their spectral properties do not fall within the useful NIR “blood window” of 700-800 nm, which facilitates analyte detection in blood. Rhodamines are “red” or long-wavelength xanthene dyes. One notable long wavelength xanthene dye is rhodamine 800 which emits at the interface of the red and NIR, a few nanometers above or below 700 nm depending on the solvent. However, it suffers from limited water solubility and dimer formation and a small Stokes shift of 16 nm (Sauer et al., J. Fluoresc., 1995, 5, 247-261), which complicates analysis in blood. Another innovation includes “JA” dyes which shift the spectra toward longer wavelength through the addition of double bonds to the nitrogen-containing rings. (Sauer et al.; U.S. Pat. No. 5,750,409). Arden-Jacob and co-workers developed an improved series of fluorophores for biodiagnostics in the red region. However, these dyes exhibit rather small Stokes shifts and do not absorb or emit in the NIR (U.S. Pat. No. 5,750,409).
Annulation is another approach used to produce longer wavelength fluorophores. Type [c] annulated xanthenes include seminaphthofluorescein (SNAFL) and seminaphthorhodafluor (SNARF) compound developed by Haugland (Whitaker et al., Anal. Biochem., 1991, 194, 330-344), which have been used as ratiometric pH sensors, metal ion sensors and imaging probes. (Chang et al., PNAS, 2004, 101, 1129-1134; Nolan et al. J. Am. Chem. Soc., 2007, 129, 5910-5918.)
The detection of biologically important thiols has been the focus of much research. Different naturally-occurring thiols, which may have similar structures, may have quite different physiological properties. The physiological effects and correlations that have been observed for these thiols are a public health concern. Examples of low molecular weight thiols that have more-or-less similar structures, but that have disparate physiological properties, include cysteine (Cys), homocysteine (Hcy), glutathione (GSH), N-acetylcysteine, and penicillamine.
Glutathione is of particular interest to medical professionals. Glutathione levels are indicative of oxidative stress. Additionally, low glutathione levels may be linked, for example, to mitochondrial diseases, autism, and mercury poisoning.
Thiols are easily oxidized, and are typically colorless and non-fluorescent at visible wavelengths. Most reported methods for thiol detection have been based upon nonspecific redox chemistry, immunoassays, or upon derivatization with chromophores or fluorophores. Generic methods for detecting thiols do not readily distinguish among the structurally similar species. There is a substantial need for improved methods for detecting and quantifying biological thiols.
Methylviologen (MV2+) is a 4,4′-dipyridyl dication:
MV2+ has been used as an oxidant in an investigation of the equilibrium kinetics of both the reducing disulfide and the α-amino carbon-centered radicals derived from Hcy, Cys and GSH. Reducing radical formation was monitored via changes in the UV-Vis spectra of solutions containing the methylviologen radical cation that formed in the presence of the biological thiols. See R. Zhao et al., “Kinetics of one-electron oxidation of thiols and hydrogen abstraction by thiyl radicals from α-amino C—H bonds,” J. Am. Chem. Soc., vol. 116, pp. 12010-12015 (1994); and R. Zhao et al., “Significance of the intramolecular transformation of glutathione thiyl radicals to α-aminoalkyl radicals. Thermochemical and biological implications,” J. Chem. Soc., Perkins Trans., vol. 2, pp. 569-574 (1997) It was surmised that formation of the reducing α-aminoalkyl radical derived from Hcy should be particularly favorable, due to an intramolecular hydrogen abstraction mechanism involving a five-atom ring transition state (See FIG. 1A). By contrast, in the case of either Cys or GSH, H-atom abstraction to a reducing carbon-centered radical would proceed via less-favored four-membered ring (FIG. 1B) or nine-membered ring (not shown) transition state geometries, respectively. See FIGS. 1A and 1B, depicting the inferred proton abstraction leading to formation of the α-aminoalkyl radical from the thiyl radicals of Hcy and Cys, respectively. These references do not disclose any appreciable colorimetric selectivity among homocysteine, cysteine, and glutathione.
U.S. Publication 2008/0261315, which is incorporated herein by reference, discloses a method for selectively determining homocysteine with methylviologen. Heating a sample containing Hcy with a colorless solution of methylviologen for five minutes or longer at a temperature between about 25° C. and 110° C. and a pH between about 3.9 and about 9.5 produces a visible color change. Color formation can be monitored via the appearance of absorption peaks at 398 nm and 605 nm. In contrast, samples containing Cys or GSH remain colorless when heated with a solution of methylviologen under similar conditions.